P25a ⁄ TPPP expression increases plasma membrane presentation of the dopamine transporter and enhances cellular sensitivity to dopamine toxicity Anja W Fjorback1,2,*, Sabrina Sundbye2, Justus C Dachsel2, , Steffen Sinning1, Ove Wiborg1 ă and Poul H Jensen2 Centre for Psychiatric Research, Aarhus University Hospital, Denmark Department of Medical Biochemistry, Aarhus University, Denmark Keywords dopamine transporter; p25a; Parkinsons disease; toxicity; TPPP; tubulin polymerization promoting protein Correspondence P.H Jensen, Department of Medical Biochemistry, Aarhus University, Ole Worms Alle 1170, DK-8000 Aarhus C, Denmark Fax: +45 86131160 Tel: +45 89422856 E-mail: phj@biokemi.au.dk *Present address Stereology and EM Research Laboratory, Aarhus University, Denmark  Present address Division of Neurogenetics, Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL 32224, USA (Received 21 June 2010, revised November 2010, accepted 20 November 2010) doi:10.1111/j.1742-4658.2010.07970.x Parkinson’s disease is characterized by preferential degeneration of the dopamine-producing neurons of the brain stem substantia nigra Imbalances between mechanisms governing dopamine transport across the plasma membrane and cellular storage vesicles increase the level of toxic pro-oxidative cytosolic dopamine The microtubule-stabilizing protein p25a accumulates in dopaminergic neurons in Parkinson’s disease We hypothesized that p25a modulates the subcellular localization of the dopamine transporter via effects on sorting vesicles, and thereby indirectly affects its cellular activity Here we show that co-expression of the dopamine transporter with p25a in HEK-293-MSR cells increases dopamine uptake via increased plasma membrane presentation of the transporter No direct interaction between p25a and the dopamine transporter was demonstrated, but they co-fractionated during subcellular fractionation of brain tissue from striatum, and direct binding of p25a peptides to brain vesicles was demonstrated Truncations of the p25a peptide revealed that the requirement for stimulating dopamine uptake is located in the central core and were similar to those required for vesicle binding Co-expression of p25a and the dopamine transporter in HEK-293-MSR cells sensitized them to the toxicity of extracellular dopamine Neuronal expression of p25a thus holds the potential to sensitize the cells toward dopamine and toxins carried by the dopamine transporter Structured digital abstract l MINT-8055798: DAT (uniprotkb:Q01959) and p25 alpha (uniprotkb:O94811) colocalize (MI:0403) by fluorescence microscopy (MI:0416) l MINT-8054201: p25 alpha (uniprotkb:B1Q0K1), bip (refseq:GI:194033595), Synaptophysin (uniprotkb:Q62277), Alpha-synuclein (uniprotkb:Q3I5G7) and DAT (uniprotkb:C6KE31) colocalize (MI:0403) by cosedimentation in solution (MI:0028) l MINT-8055878: Synaptophysin (uniprotkb:Q62277), bip (refseq:GI:194033595) and p25-alpha (uniprotkb:O94811) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029) Abbreviations a-syn, a-synuclein; DA, dopamine; DAT, dopamine transporter; NET, norepinephrine transporter; PBSCM, phosphate-buffered saline supplemented with Ca and Mg; PD, Parkinson’s disease; SERT, serotonin transporter; VMAT-2, vesicle monoamine transporter-2 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 493 p25a regulates dopamine transporter function A W Fjorback et al Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is characterized by motor dysfunctions, including resting tremor, postural imbalance, slowness of movement and muscle rigidity The motor dysfunctions are preferentially caused by loss of dopamine (DA)-producing neurons in the substantia nigra pars compacta, which results in a depleting of DA in their projection area in the striatum [1,2] The loss of dopaminergic neurons plays an essential role in causing motor dysfunction, as demonstrated by complete reversal of the symptoms in newly diagnosed PD patients when treated with DA receptor agonists or the DA precursor l-Dopa (l-3,4-dihydroxyphenylalanine) [3–6] DA is a neurotransmitter that is released into the synaptic cleft by fusion of DA storage vesicles with the plasma membrane DA is then transported back into the pre-synaptic neuron via the plasma membrane DA transporter (DAT) The cytosolic level remains low, as DA is subsequently transported into storage vesicles by the vesicle monoamine transporter-2 (VMAT-2) [7,8] However, the selective vulnerability of dopaminergic neurons is hypothesized to be caused by oxidative stress produced by DA metabolism Normal catabolism of DA by monoamine oxidase generates hydrogen peroxide that can be broken down into highly reactive hydroxyradicals in the presence of iron [3,4] The cytosolic DA represents an additional risk of oxidative stress, as DA can auto-oxidize at the neutral pH of the cytosol and thereby form toxic DA-quinone species, superoxide radicals and hydrogen peroxide The low pH in the DA storage vesicles stabilizes DA against oxidative breakdown Thus it is important to maintain a low cytosolic concentration of DA The low level of DA is thought to be maintained by a regulated balance between the functional levels of plasma membrane DAT and vesicular VMAT-2 The protein a-synuclein (a-syn) is known to play a central role in PD because mutations in its gene cause familial PD, and it is a major component of Lewy bodies in the sporadic forms of the disease [9] All though little is known about events that triggers the death of dopaminergic neurons, it has been shown that a-syn affects cellular DA homeostasis as cellular a-syn decreases DAT-mediated DA uptake via mechanisms inhibited by PD-causing mutations [10,11] Because of the a-syn-mediated effect on DA uptake, abnormal function or aggregation of a-syn in dopaminergic neurons may affect the DA balance, and thereby increase oxidative stress in the cell The brain-specific protein p25a is normally only expressed in oligodendrocytes, where it is thought to 494 affect myelin metabolism [12], possibly via modulation of microtubule dynamics [13] However, p25a is abnormally expressed in neurons in a range of neurodegenerative diseases, where it is found in the cytoplasm and nucleus and is often associated with inclusions containing aggregated a-syn [12,14–16] It has been shown that p25a directly stimulates the aggregation of a-syn, and thereby induces cytotoxicity [16,17] We hypothesized that expression of p25a in dopaminergic neurons, in addition to its putative effects on a-syn aggregation, may increase the sensitivity to dopaminergic stress, thus contributing to cell vulnerability We demonstrate that p25a increases DAT-mediated DA uptake in transiently transfected HEK-293-MSR cells, an increase that is caused by elevated DAT expression at the cell surface This increase in DA uptake subsequently leads to increased sensitivity towards cellular DA No direct interaction between DAT and p25a was demonstrated, but both co-fractionated with light vesicle fractions from porcine striatum, and direct binding of recombinant p25a protein to brain vesicles was demonstrated for the first time Abnormal p25a expression in dopaminergic neurons thereby has the potential to increase the sensitivity to DA-dependent oxidative stress in PD via actions on DA-containing vesicles Results p25a stimulates the membrane expression of DAT A potential effect of p25a on DAT function was addressed by expressing DAT alone and in the presence of p25a in HEK-293-MSR cells To validate the specificity of the C-20 goat polyclonal IgG, against C-terminal cytoplasmic domain of DAT, HEK-293-MSR cells were either transfected with the DAT pcDNA.3.1 vector or the empty control vector for 48 h before analysis by immunoblotting (Fig 1A) and immunofluorescence microscopy (Fig 1B) The immunoblot analysis showed the presence of a strong broad band centered around 75 kDa in the DAT-expressing cells that was absent in the mock-transfected cells The broad band is probably due to the presence of glycosylated and non-glycosylated DAT species The mock-transfected cells lacked DAT-reactive bands but exhibited a weak immunoreactive smear at  150 kDa that was also faintly visible for DAT-expressing cells The immunofluorescence analysis demonstrated strong DAT immunoreactivity on the plasma membrane of the DAT-expressing cells, with essentially no signal for the mock-transfected control FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS A W Fjorback et al A C D E p25a regulates dopamine transporter function B Fig p25a increases dopamine uptake by increasing the surface expression of the dopamine transporter (A) HEK-293-MSR cells were transiently transfected with DAT expression vector or empty control vector for 48 h, and extracted for immunoblot analysis Protein aliquots (20 lg) were resolved by 8–16% SDS ⁄ PAGE followed by electroblotting The filter was first probed with the C-20 goat antibody, after which the membrane was stripped and reprobed with an anti-actin antibody Top panel, anti-DAT immunoreactivity; molecular size markers indicated on the left Lower panel, actin immunoreactivity demonstrating equal loading Lanes marked by DAT and MOCK represent cells transfected with DAT expression vector and empty control vector (B) Cells transfected as in (A) were analyzed by confocal laser scanning microscopy using the C-20 antibody Left panels show anti-DAT immunoreactivity and right panels show phase contrast images of the same cells Top row, transfected with empty vector; lower row, transfected with DAT vector (C) Uptake of increasing concentrations of 3[H]-DA was measured in HEK-293-MSR cells transiently transfected with DAT and p25a, a-syn or the empty vector as a negative control The x axis shows the total concentration of DA and the y axis shows the DA uptake after 10 The y axis demonstrates specific uptake, defined as total uptake subtracted the uptake in the presence of the DAT inhibitor cocaine (200 lM) and represents the mean ± SEM of four experiments Uptake in vector control transfected cells was determined for lM dopamine and found to be negligible (open square) The effect of p25a on DAT-mediated DA uptake was compared to that in the presence of the empty vector control, and was found to be different at Vmax values (*P < 0.05, Student’s t test), as was the a-syn-mediated decrease in DAT uptake at Vmax (*P < 0.05, Student’s t test) (D) Expression of p25a and a-syn affects surface but not total DAT expression HEK-293MSR cells transiently transfected with DAT and p25a, a-syn or the empty vector were either extracted directly in lysis buffer or subjected to cell surface biotinylation with EZ-Link Sulfo-NHS-SS-Biotin followed by extraction This cross-linker allows cleavage between biotin and the target by reducing the disulfide bridge The biotinylated proteins were captured by incubating with NeutraAvidin beads The total and surface-bound protein fractions were analyzed by reducing SDS ⁄ PAGE followed by Western blotting with antibodies toward DAT, p25a, a-syn and b-actin (as loading control) Total cellular DAT is present as two bands representing glycosylated and non-glycosylated DAT, and was slightly lower in the double transfected cells compared to those expressing DAT alone By contrast, surface-bound DAT was increased in p25a-expressing cells and decreased in a-syn-expressing cells compared to control-transfected cells (E) Quantification of the surface DAT bands in (B) normalized against b-actin as analyzed by densitometry and shown as a percentage of the control The columns represent means ± SEM of three experiments Comparison of the three conditions by one-way ANOVA was significant (P < 0.05) Individual comparison of p25a or a-syn to control by Student’s t test was also significant (*P < 0.05) (Fig 1B) Hence, the C-20 antibody binds specifically and selectively to DAT in our cellular system, in agreement with previous reports [18] Figure 1C shows that expression of DAT caused a cocaine-inhibitable and saturable uptake of [3H]-DA in HEK-293-MSR cells, and this was enhanced by co-expression with p25a As a control, we also FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 495 p25a regulates dopamine transporter function A W Fjorback et al co-expressed DAT with a-syn, which is known to decrease DAT activity [10], and noted a decrease in DAT-mediated uptake Kinetic analysis showed that p25a increased the Vmax without affecting the Km (Table 1) This effect was not caused by increased DAT expression, as total cellular extracts showed a minor decrease in DAT expression as determined by C-20 antiDAT IgG binding when co-expressed with either p25a or a-syn, compared to the empty control vector (Fig 1D, top row) Biotinylation of surface proteins was performed to determine whether the increased Vmax represents a redistribution of DAT from intracellular vesicles to the plasma membrane HEK-293-MSR cells expressing DAT alone and in the presence of p25a or a-syn were incubated with the membrane-impermeable cross-linker EZ-Link Sulfo-NHS-SS-Biotin, followed by detergent extraction and isolation of biotinylated protein by incubation with streptavidin-coated beads The second row of Fig 1D shows that the level of biotinylated plasma membrane DAT was highest in p25a-expressing cells Quantification of the data showed that p25a stimulated a significant ( 50%) increase in plasma membrane DAT, in contrast to the significant decrease of  50% when co-expressed with a-syn (Fig 1E) Therefore, p25a stimulates cellular DA uptake by translocating intracellular DAT to the plasma membrane strate a folded central core and unfolded N- and C-terminal extensions of  45 and 70 amino acid residues, respectively We constructed two deletion mutants: p25aDN, which lacked N-terminal residues 3–43, and p25aDC, which lacked C-terminal residues 156–219 (Fig 2A) The truncated p25a proteins were compared with full-length p25a with respect to their ability to induce increased DA uptake when co-expressed with DAT Figure 2B demonstrates that the full-length and two truncated p25a proteins all induce a similar increase in DA uptake when expressed in the HEK293-MSR cells expressing DAT alone This suggests that the central folded core domain is responsible for the stimulatory effect on DAT DAT belongs to the family of neurotransmitter transporters that includes the norepinephrine and serotonin transporters (NET and SERT), which share a range of structural characteristics The expression of a-syn has been shown to decrease the membrane expression of all three transporters [10,23,24] Table shows that p25a selectively stimulates uptake via DAT A Structural requirements for p25a-stimulated dopamine uptake p25a has a folded dynamic structure [19], and NMR spectroscopic data of p25a homologs from mice, Caenorhabditis elegans and humans [20–22] demonTable Determination of Vmax and Km for DAT, NET and SERT in the presence and absence of p25a HEK-293-MSR cells were transiently transfected with DAT, NET or SERT and with p25a or mock vector The cells were subsequently used for analysis of DA and serotonin uptake, respectively 3[H]-DA was used to determine Vmax curves for DAT and NET, as 3[H]-DA can also be used by NET, and [H]-serotonin was used for determination of Vmax curves for SERT The Vmax curve was obtained by 10 incubation with 3[H]-DA and 3[H]-serotonin diluted 20 times with unlabeled DA and serotonin, respectively, at eight concentrations ranging from to 10 lM Vmax and Km values are given as means ± SEM (n = 3) Vmax (pmolỈmin)1 per well) SERT ⁄ pcDNA3 SERT ⁄ p25a NET ⁄ pcDNA3 NET ⁄ p25a DAT ⁄ pcDNA3 DAT ⁄ p25a 496 Km (lM) 0.648 0.588 0.157 0.135 0.591 0.792 3.2 3.81 1.25 1.85 2.49 2.47 ± ± ± ± ± ± 0.04 0.06 0.01 0.007 0.013 0.016 ± ± ± ± ± ± 1.3 0.96 0.14 0.46 0.80 0.63 B Fig Effect of p25a deletions on dopamine uptake (A) p25a contains a folded central core (gray box) and unfolded termini Expression vectors for deletion mutants p25aDN, lacking residues 3–43, and p25aDC, lacking residues 156–219, were constructed (B) [3H]DA (2.5 lM) uptake in HEK-293-MSR cells transfected with DAT and control vector or DAT in combination with wild-type p25a, p25aDM or p25aDC vector Bars represent mean ± SEM of three independent experiments performed in triplicate, and are normalized against a control expressing DAT alone Comparison of the four conditions by one-way ANOVA indicates significant differences (P < 0.05) *P < 0.05 as compared to control by Student’s t test FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS A W Fjorback et al but not via SERT and NET Hence, the central folded core of p25a selectively increases the surface expression of DAT p25a regulates dopamine transporter function A p25a and DAT are associated with light brain vesicles Immunoprecipitations and subcellular fractionations were performed to identify a possible interaction between p25a and DAT Immunoprecipitations in cellular detergent extracts previously demonstrated an interaction between a-syn and DAT [10], but we were unable to co-immunoprecipitate p25a and DAT (data not shown) A series of protocols and antibodies were tested for co-immunoprecipitating p25a with DAT and DAT with p25a, and various antibodies were tested for precipitation of DAT, including epitope-specific V5 antibody for a tagged DAT fusion protein DAT is an integral membrane protein, so we tested whether p25a was present in subcellular brain fractions enriched in DAT-containing vesicles We performed the subcellular fractionation on tissue from the nucleus caudatus of the porcine striatum because this tissue is enriched in dopaminergic nerve terminals Figure 3A shows that DAT was highly enriched in the light fraction LP2, together with the pre-synaptic vesicle marker synaptophysin Lower concentrations of DAT were present in the vesicular fractions P3 and LP1, together with the endoplasmic reticulum marker, the 78 kDa glucose regulated protein/BiP (GRP78) BIP p25a was primarily localized in two fractions (S3 and LP2), with minor amounts in the remaining fractions Its presence in the cytosolic fraction S3 was expected as it has hitherto been considered a soluble cytosolic constituent The even larger p25a concentration in the light vesicular LP2 fraction, together with DAT, was unexpected and suggests that p25a may have previously unknown functions related to vesicular biology For comparison, the presence of a-syn was also investigated because this protein is known to be both cytosolic and vesicle-associated a-syn showed a less distinct localization, being present in most fractions in significant concentrations For instance, when the synaptosomal fraction P2 is subjected to hypotonic lysis, a-syn appear in the cytosolic fraction LS2 and the vesicular fraction LP2 in almost equal amounts By contrast, p25a appears almost exclusively in the light vesicular fraction LP2, with no protein in the cytosolic fraction LS2 This indicates some specificity in vesicle binding when compared to the membraneassociated proteins DAT and synaptophysin Having established for the first time that p25a is a vesicle-associated protein in subcellular brain fractions, B Fig Co-fractionation of DAT and p25a in subcellular fractions of porcine striatal tissue and localization in cells (A) Isolated porcine striatal tissue (nucleus caudatus) was fractionated as described in Experimental procedures The fractions were crude pellet (P1), microsomal fraction (P3), cytosolic fraction (S3), lysed dense synaptosomal pellet (LP1), lysed light synaptosomal pellet (LP2) and lysed synapsomal cysosol (LS2) Protein aliquots (20 lg) for each fraction were subjected to SDS ⁄ PAGE followed by immunoblotting; the membrane was probed using antibodies against BIP (endoplasmic reticulum marker), DAT, synaptophysin (synaptic vesicle marker), p25a and a-synuclein Molecular size markers are shown on the left, and the antigens are indicated on the right (B) Localization of DAT and p25a transiently expressed in SH-SY5Y cells was analyzed by confocal laser scanning microscopy A representative cell is shown with granular and membrane staining of DAT and more diffuse cytoplasmic p25a staining To better visualize the possible co-localization of p25a and DAT, the neurites indicated by the inset in the lower left panel are enlarged in the lower right panel It is evident that some co-localization occurs, although this is not complete we investigated whether purified recombinant p25a can bind directly to brain vesicles A vesicle-binding experiment comprising a vesicle-flotation assay was used, as described previously for a-syn [25], wherein brain homogenate was supplemented with 55% sucrose, overlaid with a sucrose density gradient, and subjected to ultracentrifugation Lipid-containing vesicles have a low density and float up into the gradient, whereas FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 497 p25a regulates dopamine transporter function A W Fjorback et al cytosolic proteins remain in the dense bottom fractions Figure 4B shows that endogenous p25a is distributed in both the dense cytosolic bottom fraction and the light vesicle-containing fractions, in agreement with the subcellular fractionation into vesicular and cytosolic fractions (Fig 3A) For the binding analysis, we used the following recombinant human p25a peptides: full-length p25 and truncated p25aDN and p25aDC (Fig 4A) The purified p25a core peptide corresponding to residues 44–156 was highly insoluble except in 10 mM acetic acid However, its insolubility in the neutral binding buffers made it impossible to study in the binding assay (data not shown) The full-length p25a protein was subsequently biotinylated to allow it to be distinguished from endogenous porcine brain p25a Incubation of brain vesicles with biotinylated p25a prior to vesicle flotation resulted in the recovery of biotinylated p25a in light fractions 2–6 As a control, a sample was supplemented with 1% Triton X-100 and 1% SDS to A B Fig p25a binds to porcine brain vesicles (A) The wild-type and deletion constructs of p25a p25aDN, p25aDC and the p25a core, corresponding to residues 44–156, were cloned into pET11d vector, expressed in E coli and purified The purified proteins were subjected to reducing SDS ⁄ PAGE and Coomassie blue staining Molecular size markers are shown on the left (B) The association of p25a with brain vesicles was demonstrated by subjecting a porcine brain homogenate to a vesicle flotation assay The assay is based on the light vesicles floating in the density gradient and the cytosolic proteins remaining in the bottom fractions The homogenate was supplemented with sucrose to 55%, overlaid with a sucrose density gradient of 48–20%, and subjected to ultracentrifugation, after which nine fractions were isolated from the top of the gradient The isolated fractions were subjected to reducing SDS ⁄ PAGE and immunoblotting for detection of endogenous p25a (E-p25a), BIP and synaptophysin BIP and synaptophysin immunoreactivity were detected on the filter where biotinylated p25a (B-p25a) had been resolved To ensure that the flotation was due to binding to light vesicles, 1% Triton X-100 and 1% SDS were added to dissolve the membranes, as demonstrated for BIP, synaptophysin and biotinylated p25a (B-p25a + TX) As a positive control for the immunoblot, purified recombinant p25a is shown on the left (R-p25a) To measure direct binding of recombinant full-length p25a to the brain vesicles, purified protein was biotinylated (B-p25a) and incubated with the brain homogenate prior to vesicle flotation, followed by visualization with horseradish peroxidase-conjugated streptavidin Similarly, the truncated peptides p25aDN and p25aDC were incubated with the homogenate and treated like B-p25a, except they were detected with using p25a-1 antibody Representative data from one of three independent experiments are presented 498 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS A W Fjorback et al dissolve the vesicles prior to analysis This caused the biotinylated p25a tracer to remain in the bottom fractions, thus demonstrating that the flotation was due to binding to vesicles Because the vesicle-binding profile of biotinylated p25a differed from that of the endogenous p25a, the filter used for biotinylated p25a was probed for the ER marker BIP and the synaptic vesicle marker synaptophysin Both showed a profile essentially identical to that of biotinylated p25a, thus confirming its vesicle-binding properties Moreover, solubilization of the vesicles caused BIP, synaptophysin and biotinylated p25a to remain in the bottom fractions, thus confirming that vesicle binding was responsible for their flotation The electrophoretic mobility of the two truncated p25a species differed from that of full-length p25a (Fig 4A), so biotinylation was not needed for their detection by immunoblotting Figure 4B shows that both truncated proteins were distributed in the density gradient similar to the endogenous p25a Detergent treatment completely abrogated their flotation (data not shown) as demonstrated for the biotinylated p25a This indicates the termini are dispensable for vesicle binding and suggests a critical role for the core domain To visualize the putative cellular co-localization of DAT and p25a, laser scanning confocal microscopic imaging of SH-SY5Y cells transiently transfected with DAT and p25a was performed (Fig 3B) SH-SY5Y cells were chosen because they have a more flattened morphology, with several thin processes, thus allowing improved visualization of subcellular co-localization compared to the epithelial morphology of HEK-293MSR cells DAT was found to localize in the cellular processes, together with granular intracellular staining compatible with an association with vesicles and the plasma membrane p25a showed cytosolic staining, was localized in the cytosol with a less granular appearance than DAT Focusing on the finer neurites revealed that p25a and DAT co-localize in some instances, but it should be remembered that both proteins are highly over-expressed, and this may saturate specific interactions and reduce the signal-to-noise ratio (Fig 3B) Therefore, we conclude that p25a is a brain vesiclebinding protein that may associate with DAT-containing brain vesicles p25a increases dopamine toxicity Oxidative stress caused by cytoplasmic DA may contribute to PD-associated cell death To investigate a putative role for aberrant neuronal p25a expression in this scenario, we incubated HEK-293-MSR cells with 0.5 mm DA for 24 h, and quantified their viability p25a regulates dopamine transporter function using the MTT assay Expression of DAT, p25a and a-syn alone, or p25 or a-syn together with DAT respectively, did not affect survival (data not shown), but the presence of 0.5 mm DA for 24 h caused a 20% reduction in the survival of DAT-expressing cells but had no effect on p25a- and a-syn-expressing cells (Fig 5) Co-expression of DAT and p25a enhanced the DA toxicity to 50%, in agreement with an increased DA uptake (Fig 5) By contrast, co-expression of DAT with a-syn was used as a control for protein over expression a-syn eliminated the toxicity produced by addition of DA (Fig 5) Discussion Degeneration of the dopaminergic neurons of the substantia nigra pars compacta is responsible for the major motor symptoms of PD The neurotransmitter DA has been hypothesized to play a major role in this selective loss of dopaminergic neurons, and the cytosolic fraction of DA is considered particularly toxic [3,7,26] This highlights the importance of a controlled balance between the two transport systems that regulate cytosolic DA: the DAT, which transports Fig Expression of p25a increases DAT-mediated DA toxicity HEK-293-MSR cells transfected with DAT + empty vector, p25a + empty vector, a-syn + empty vector, DAT ⁄ P25a and DAT ⁄ a-syn to obtain equal concentrations of vector DNA were incubated in the absence and presence of 0.5 mM DA for 24 h and subjected to analysis of viability by the MTT assay Co-transfection of DAT with empty vector served as a control for background sensitivity to DA toxicity in the absence of p25a Displayed is the percentage survival of DA-treated cells relative to cells not treated with dopamine Cells not expressing DAT were insensitive to DA, but DAT expression caused a minor but significant loss of survival Co-expression of DAT and p25a increased this cell loss, but co-expression with AS attenuated it Bars represents means ± SD of triplicates in one representative experiment of three performed, except for AS, which was only included in two experiments *P < 0.05 compared to controls (Student’s t test) FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 499 p25a regulates dopamine transporter function A W Fjorback et al extracellular DA into the cytosol, and VMAT-2, which transports cytosolic DA into storage vesicles The critical role of these systems in degeneration of dopaminergic neurons has been shown through both genetic and chemical evidence DAT is an import molecule for environmental toxins causing PD [27], and there is an increased sensitivity toward DAT-dependent toxins in VMAT-2 heterozygous mice [28] The instability of DA at the neutral pH of the cytosol causes the formation of toxic aminochromes, and these species are not formed when DA is protonated at the acidic pH of the storage vesicles Factors that increase the surface expression of DAT holds the potential to increase cytosolic DA by disturbing the balance between DAT and VMAT-2 and causing chronic oxidative stress due to an increased concentration of toxic DA metabolites [26] Abnormal expression of p25a in nerve cells has been shown in the Parkinson’s disorders PD, multiple systems atrophy and Lewy body dementia [16,29,30], in which it is principally associated with a-syn-containing inclusions but also with other a-syn-negative cytoplasmic and nuclear inclusions [14] The p25a protein is normally expressed in myelinating oligodendrocytes [12,31] p25a is a microtubuleassociated protein that stimulates the aggregation of tubulin in vitro [13], but cellular experiments have also shown an effect on the actin system via (LIMK1) a cytoplasmic serine/threonine kinase [32] The importance of vesicular transport in regulating DAT sorting to the plasma membrane, combined with ectopic expression of the microtubule regulator p25a in degenerating dopaminergic neurons, suggested that p25a could be a regulator of DAT activity and a contributing factor in sporadic parkinsonistic syndromes We have shown that co-expression of DAT with p25a in HEK-293-MSR cells increased DA uptake compared to controls via an increased plasma membrane presentation of DAT, and this sensitizes the cells to DA-mediated toxicity Hence, pathological expression of p25a in DAT-expressing neurons in PD may potentiate the toxic effects of dopamine Here we show for the first time that p25a binds to brain vesicles, and demonstrate co-fractionation of p25a with DAT-containing vesicle fractions By contrast, no direct interaction with DAT could be demonstrated by immunoprecipitation, as was previously demonstrated between a-syn and DAT [33,34] To investigate the co-fractionation, we used porcine striatal tissue from the nucleus caudatus that are innervated by dopaminergic axonal projections from the substantia nigra and which are rich in DAT Surprisingly, the highest concentration of p25a was present in the LP2 light synaptosomal vesicles, which were also enriched in DAT and synapto500 physin However, whereas DAT and synaptophysin were both enriched in vesicle-containing fractions P3, LP1 and LP2, p25a was predominantly present in LP2 The second major location of p25a was in the cytosolic S3 fraction, and only minor amounts of p25a were present in the other fractions The partial co-fractionation was confirmed by confocal laser scanning microscopical detection of human p25a and DAT transiently expressed in SH-SY5Y cells, showing partial overlap of the two antigens We performed a vesicle binding experiment to confirm that the association of p25a with brain vesicles could be mediated by direct binding, and also to investigate structural requirements for an association The assay is based on subjecting a brain homogenate to density gradient centrifugation, whereby the lower density of vesicles makes them float in the gradient whereas soluble proteins remain at the bottom Biotinylated fulllength p25a did bind to vesicles, and addition of detergents to solubilize the vesicles abolished the flotation of biotinylated p25a Non-biotinylated N- and C-terminally truncated p25a proteins showed a vesicular binding pattern similar to that of endogenous p25a, suggesting that the folded core domain possesses the structure necessary for the vesicle binding function However, direct evidence could not be obtained because the recombinant p25a core protein was insoluble at neutral pH The similarity between the core sequences of the a, b and c p25 gene products suggests that the vesicle-binding function may be a common property for this protein family [35] It should be remembered that p25a is preferentially expressed in oligodendrocytes, and is present in both the cell body and myelin sheets [12,31], and its putative functions related to microtubules and vesicle transport warrant further investigations The specificity of vesicle association in combination with the direct binding of p25a to vesicles suggest the presence of vesicular p25a receptors, and this is now under investigation The functional role of the vesicle interaction was confirmed by cellular DA-uptake experiments, because both the N- and C-terminally truncated p25a proteins stimulated DA uptake to the same extent as the fulllength p25a when co-expressed with DAT in HEK293-MSR cells The selective effect of p25a on DAT activity but not NET and SERT may reflect the fact that DAT is present in specific sorting vesicles that are targeted by p25a, and thus resembles the apical DAT sorting in MDCK cells, in contrast to the basolateral sorting of NET and SERT in these cells [36] This interpretation is corroborated by the increased sensitivity to dopamine of stable SH-SY5Y clones expressing human p25a However, variability in DAT expression due to clonal effects not attributed to the transgene FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS A W Fjorback et al cannot be excluded, because anti-DAT immunoblot analyses failed to detect the low endogenous level We conclude that p25a has vesicle-binding properties and facilitates cellular DA uptake via increased plasma membrane presentation of DAT Determination of the biological role of the vesicle binding and its potential role in relation to PD pathogenesis requires further experimentation, but may be exploited in creating PD models in which p25a expression in DA neurons sensitizes them to oxidative damage and prepare them for environmental toxins that use DAT for cellular entry Experimental procedures Plasmids and antibodies pcDNA3 vectors expressing DAT and NET were a kind gift from Dr Susan Amara (Center for Neuroscience, University of Pittsburgh, PA, USA) SERT was cloned into the pcDNA3 vector as previously described [37] cDNA encoding a-syn or p25a was cloned into the pcDNA3 vector as described previously [16,38] Prokaryotic expression vectors containing inserts for human p25a, N-terminally truncated by amino acid residues 3–43 (p25aDN) and C-terminally truncated by residues 156-219 (p25aDC) were used, and were purified as described previously [17,19,22,39] The central folded core of p25a, corresponding to amino acid residues 44–156, was amplified and tagged with six histidine residues by PCR using forward primers 5¢-CACCATCACGGAGCATCCCCTGAG3¢, 5¢-TCGCATCACCATCACCATCACGGAGCA-3¢ and 5¢-CACCCATGGGATCGCATCACCAT-3¢, and reverse primer 5¢-CACGGATCCCTACGTCACCCCTGA-3¢ After digestion with NcoI and BamHI restriction enzymes, the insert was ligated into pET11d vector (Novagen, Rodovre, Denmark) Correct insertion was verified by DNA sequencing (Eurofins-MWG, Martinsried, Germany) The protein was expressed in Escherichia coli BL21 (DE3) cells (Stratagene, La Jolla, CA, USA), and extracted by sonication on ice in 50 mm NaH2PO4, pH 7.0 Cell debris was removed by centrifugation at 1000 g and the supernatant was filtrated The hexahistidine-tagged p25a core protein was purified using a TALON metal affinity resin (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions The eukaryotic full-length human p25a expression vector has been described previously [17] The eukaryotic p25aDN and p25aDC vectors were constructed by PCR using cDNA coding for human p25a and primers 5¢-CACTCTAGACCATGGCTGCATCCCCTGAGCTCAGT-3¢ and 5¢-CACGGATCCCTACTTGCCCCCTTGCAC-3¢ for P25aDN, and 5¢-CACTCTAGACCATGGCTGACAAGG-3¢ and 5¢-CACGGATCCCTACGTCACCCCTGA-3¢ for P25aDC p25a regulates dopamine transporter function These fragments were then inserted into pcDNA3.1 ⁄ Zeo()) vector (Invitrogen, Carlsbad, CA) using XbaI and BamHI restriction enzymes (New England Biolabs, Denmark) Correct insertion was verified as stated above Rabbit polyclonal anti-a-syn ASY1 antibody and polyclonal rabbit anti-p25a1 were all homemade in our laboratory [16,40] Polyclonal goat anti-DAT antibody (C-20) and horseradish peroxidase-conjugated secondary anti-goat and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) The monoclonal mouse anti-b-actin antibody (clone AC15) was purchased from Sigma-Aldrich (Denmark) The mouse monoclonal anti-synaptophysin IgG was purchased from DAKO (Glostrup, Denmark) Mouse monoclonal anti-BIP IgG against the endoplasmic reticulum marker glucose-regulated protein 78 (BIP) was purchased from BD Transduction Laboratories (Denmark) Cell culture and transfection HEK-293-MSR cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, mm glutamine, 100 lgỈmL)1 streptomycin and 100 mL)1 penicillin at 37 °C and 5% CO2 The cells were transfected 48–72 h prior to the experiments with appropriate amounts of plasmid and Genejuice (Novagen) mixed with medium according to the manufacturer’s recommendations SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 5% FCS and 100 lg/mL pen ⁄ strep, and zeocin (25 lgỈmL)1) was added to the medium for selection of stably transfected cell lines The cells were not differentiated prior to experimentation Immunofluorescence microscopy For cellular localization studies, HEK and SH-SY5Y cells were transiently transfected with human DAT and p25a At 36 h after transfection, the cells were fixed in 4% paraformaldehyde for 10 min, and subsequently permeabilized for 30 (50 mm glycine, 0.1% Triton-X-100, mm CaCl2, mm MgCl2) After blocking for 30 with 3% BSA in NaCl ⁄ Pi, the transfected cells were stained with the respective primary antibodies, diluted in NaCl ⁄ Pi with 1% BSA for 60 at room temperature (anti-DAT, : 100; polyclonal rat anti-p25a1, : 1000) The secondary antibodies were Alexa Fluor 568 donkey anti-goat or Alexa Fluor 488 donkey anti-rat IgG conjugates (1 : 1000, Molecular Probes ⁄ Invitrogen) The cells were investigated using a Zeiss confocal LSM 510 META microscope with a 40 · NA 1.2 C-Apochromat objective (Zeiss, Jena, Germany) Western blotting Protein samples were prepared by incubation of transfected HEK-293-MSR cells with 300 lL lysis buffer [NaCl ⁄ Pi: FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 501 p25a regulates dopamine transporter function A W Fjorback et al 137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.4, supplemented mm EDTA, 1% Triton X-100, 0.1% SDS and protease inhibitor cocktail (Complete EDTA-free tablets, Roche, Denmark)] for 30 at °C under gentle shaking The cell lysate was cleared by centrifugation at 12 000 g at °C for 15 min, and the remaining supernatant was mixed with SDS sample buffer (250 mm Tris ⁄ HCl, pH 6.8, 5% SDS, 0.25% bromophenol blue, 25% glycerol) and left at 37 °C for 30 Fractions of the cell samples were analyzed by 8–16% SDS ⁄ PAGE and transferred to nitrocellulose membrane The membrane was blocked for h in 5% dry milk in TBST buffer (pH 8.0, 50 mm Tris ⁄ HCl, 150 mm NaCl, 0.5% Tween-20), and then probed overnight with primary antibody (1 : 1000, except ASY1 at : 500), followed by incubation with horseradish peroxidase-conjugated secondary antibody: anti-goat antibody (1 : 2500) or anti-rabbit antibody (1 : 1000) Proteins were visualized using an ECL Advance Western blotting detection kit (GE Healthcare, Denmark) and developed on a Kodak Image station 440 (Denmark) individual wells extracted in 0.1 m NaOH and measured using the bicinchoninic acid method DA toxicity assay Dose-dependent DA toxicity was evaluated in the 0.1–2.5 mm DA concentration range, and a concentration of 0.5 mm was used for further survival studies HEKMSR-293 cells were transfected in a solution with a fixed concentration of vector DNA (empty vector or the DAT, AS or p25a plasmids), and 5000 cells ⁄ well were plated in 96-well, flat-bottomed microtiter plates in a final volume of 200 lL of complete cell culture medium After 48 h, the cells were incubated in the absence or presence of 0.5 mm DA After 24 h of incubation at 37 °C, 5% CO2, the medium containing the DA was removed and the cells were washed in PBSCM to remove DA Cell viability was then measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) non-radioactive cell proliferation assay (Promega, Madison, WI, USA) Biotinylation of surface-expressed DAT Uptake activity Dopamine or serotonin uptake assays were performed 48 h after transfection of the cells The medium was removed and the cells were washed with NaCl ⁄ Pi (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.4) supplemented with 0.1 mm CaCl2 and mm MgCl2 (PBSCM) To distinguish total binding from non-specific binding, cells were incubated for 10 in either PBSCM alone or PBSCM containing 200 lm cocaine for inhibition of DAT or 200 lm S-citalopram for inhibition of SERT Then the cells were incubated with radioactive labeled [3H]-dopamine or [3H]serotonin, respectively For Km and Vmax determinations, the cells were incubated with increasing concentrations of [3H]dopamine or [3H]-serotonin diluted 20 times with unlabeled dopamine or serotonin, respectively The final concentration of dopamine and serotonin ranged from 0–10 lm For single Vmax determinations, only one concentration (5 lm) of [3H]dopamine diluted 20 times with unlabeled dopamine was used Washing twice with PBSCM terminated the uptake All washing steps were performed using an automated plate washer Following uptake, cells were solubilized in scintillation solution (MicroScient-20, Packard Bell, Denmark), and plates were counted in a Packard Top counter Values are the mean of six replicates The specific uptake was determined by subtracting the uptake counts in the absence of inhibitor from the uptake counts in the presence of cocaine or S-citalopram, respectively We also confirmed that no uptake occurred in mock-transfected HEK cells Assuming Michalis–Menten kinetics, the data were plotted and analyzed by a non-linear-squares curve fit (GraphPadPrism, Denmark) The amount of cellular protein per well for each condition was determined as the mean values for the contents of three 502 Surface biotinylation was performed essentially as previously described [41] HEK-293-MSR cells transfected with DAT in the presence or absence of p25a and a-syn, respectively, were grown in six-well plates and used at 80% confluence Cells were washed twice with ice-cold PBSCM, and incubated for 30 on ice with PBSCM containing mgỈmL)1 EZ-Link sulfo-NHS-S-S-biotin (Pierce, Rockford, IL, USA) After incubation with biotin, the cells were washed twice in PBSCM supplied with 100 mm glycine, and incubated for 20 to quench further cross-linking The quenching buffer was removed by washing twice in PBSCM, and the cells were then lysed in lysis buffer (PBSCM, 1% Triton X-100, 0.1% SDS) supplemented with Complete proteinase inhibitor (Roche) The cell lysate was transferred to Eppendorf vials, and centrifuged for 15 at 16 000 g and the supernatant was transferred to new vials A fraction was retained for determination of total protein The remainder of the supernatant was incubated with NeutraAvidin (Pierce, Rockford, IL, USA) beads to precipitate the biotinylated proteins The beads were washed four times in PBSCM before elution with 50 lL of 20 mm dithioerythreitol-containing SDS sample buffer, and incubated for 30 at 37 °C The dithioerythreitol reduces the disulfide bridge in the cross-linker between biotin and the target proteins, and thus allows their analysis by SDS ⁄ PAGE Subcellular fractionation of porcine striatal brain tissue Porcine brain cut in half in the saggital plane was obtained fresh from a local abattoir and immediately cooled on ice Within h, the nucleus caudatus, i.e the part of the FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS A W Fjorback et al striatum facing the lateral ventricle, was dissected and frozen in liquid nitrogen followed by storage at ) 80 °C A method originally described for rat brain tissue was used for subcellular fractionation [40] All procedures were performed on ice or at °C Frozen tissue (2 g) was thawed in 10 mL ice-cold homogenization buffer [320 mm sucrose, mm HEPES ⁄ NaOH, pH 7.4, mm EDTA, Complete proteinase inhibitor (Roche)] The brain was homogenized using a glass-Teflon Dounce homogenizer, and the homogenate was centrifuged for 10 at 1000 g The resulting pellet (P1) was frozen The supernatant (S1) was collected and centrifuged for 15 at 12 000 g, yielding S2 and P2 P2 was washed by resuspension in 8.5 mL of homogenization buffer, and re-centrifuged for 15 at 10 200 g, yielding P2¢ and S2¢ The pooled S2 and S2¢, designated S3, was centrifuged for h at 260 000 g, yielding cytosolic fraction S3 and microsomal fraction P3 P2¢, representing the crude synaptosomal fraction, was resuspended in homogenization buffer to a final volume of 0.9 mL, and 4.2 mL of ice-cold water was added and the suspension homogenized to allow hypertonic lysis The lysed synatosomes were immediately mixed with 30 lL m HEPES ⁄ NaOH buffer, pH 7.4, and incubated on ice for 30 After incubation, the lysed synaptosomes were centrifuged for 20 at 33 000 g to yield the lysate pellet (LP1) and supernatant LS1 LS1 was further fractionated by centrifugation for h at 260 000 g, yielding the supernatant LS2, containing the cytosol-enriched fraction, and the pellet LP2, which is enriched in synaptic vesicles The protein concentrations were measured using the bicinchoninic acid method (Sigma) Equal amounts of protein (20 lg) from each fraction were analyzed by Western blotting The Western blot was probed with antibodies for p25a, DAT, a-syn, the endoplasmic reticulum marker glucose-regulated protein 78 ⁄ BIP, the trans-Golgi marker TGN38 and the synaptic vesicle marker synaptophysin Vesicle binding assay for recombinant p25a proteins using flotation Recombinant p25a in NaCl ⁄ Pi, pH 8.0, was biotinylated using NHS-PEO4-Biotin (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions, to allow it to be distinguished from endogenous brain p25a The biotinylation reaction was quenched using m Tris, pH 8.2, and excess NHS-PEO4-Biotin was removed by fast desalting on a PC3.2 ⁄ 10 column (Amersham, Denmark) Vesicle isolation and binding were performed as described previously [25] Briefly, g porcine brain was homogenized in 2.5 mL of mm dithiothreitol, mm EDTA, 9% sucrose, 25 mm MES, pH 7.0, in the presence of protease inhibitors (Complete EDTA-free tablets; Roche) Nuclei and debris were removed by centrifugation at 500 g for at °C, and a crude vesicle fraction was isolated by ultracentrifugation of the supernatant at p25a regulates dopamine transporter function 100 000 g for h at °C After ultracentrifugation, the pellet was resuspended in 400 lL homogenization buffer, and 100 lL of resuspended vesicles were incubated with lm recombinant p25a protein (biotinylated p25a or unlabeled truncated p25DN or p25DC) for h at °C The truncated peptides migrate faster than endogenous p25a, and thus need no biotinylation For the negative control, 1% Triton X-100 and 1% SDS were added to the sample to solubilize the vesicles The solution was brought to 55% sucrose in a volume of 0.47 mL, and overlaid with mL 48–20% sucrose gradient The samples were then subjected to ultracentrifugation at 100 000 g for 16 h at °C The gradient was divided into nine fractions starting from the top The protein content of each fraction was precipitated with 20% trichloroacetic acid and subjected to SDS ⁄ PAGE followed by Western blotting Endogenous p25a as well as recombinant p25DN and p25DC were visualized using anti-p25a1 IgG (rabbit) (1 : 1000) followed by horseradish peroxidase-conjugated anti-rabbit IgG (1 : 1000) (DAKO) Recombinant biotinylated p25a was visualized using horseradish peroxidase-conjugated streptavidin (1 : 1000) (GE Healthcare) Statistics One-way ANOVA was performed with post hoc analysis for comparison of the means of more than two groups, with P < 0.05 considered significant Student’s t test was used to compare means between individual groups, and P values < 0.05 were considered significant Acknowledgements We thank Jette B Lauritsen for technical assistance with the SH-SY5Y cells and Anette Larsen for the artwork This work was supported by the Danish Council for Strategic Research (Histoinformatics Centre), the EU Marie Curie Molecular Neuroimmunology PhD program (MNIEST, contact number MEST-CT514333), the Danish innovation consortium CureND, the Lundbeck Foundation, the Danish Medical Research Council (271-05-0166), the Augustinus Foundation, the Familien Hede Nielsen Foundation and the HistoInformatic Center References Fearnley JM & Lees AJ (1991) Ageing and Parkinson’s disease: substantia nigra regional selectivity Brain 114, 2283–2301 Hirsch E, Graybiel AM & Agid YA (1988) Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson’s disease Nature 334, 345– 348 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 503 p25a regulates dopamine transporter function A W Fjorback et al Jenner P & Olanow CW (1996) Oxidative stress and the pathogenesis of Parkinson’s disease Neurology 47, S161–S170 Kim WG, Mohney RP, Wilson B, Jeohn GH, Liu B & Hong JS (2000) Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia J Neurosci 20, 6309–6316 Olanow CW, Jenner P & Brooks D (1998) Dopamine agonists and neuroprotection in Parkinson’s disease Ann Neurol 44, S167–S174 Smith LA, Jackson MJ, Al Barghouthy G, Rose S, Kuoppamaki M, Olanow W & Jenner P (2005) Multiple small doses of levodopa plus entacapone produce continuous dopaminergic stimulation and reduce dyskinesia induction in MPTP-treated drug-naive primates Mov Disord 20, 306–314 Vergo S, Johansen JL, Leist M & Lotharius J (2007) Vesicular monoamine transporter regulates the sensitivity of rat dopaminergic neurons to disturbed cytosolic dopamine levels Brain Res 1185, 18–32 Zheng G, Dwoskin LP & Crooks PA (2006) Vesicular monoamine transporter 2: role as a novel target for drug development AAPS J 8, E682–E692 Goedert M, Spillantini MG & Davies SW (1998) Filamentous nerve cell inclusions in neurodegenerative diseases Curr Opin Neurobiol 8, 619–632 10 Wersinger C, Prou D, Vernier P & Sidhu A (2003) Modulation of dopamine transporter function by a-synuclein is altered by impairment of cell adhesion and by induction of oxidative stress FASEB J 17, 2151–2153 11 Wersinger C & Sidhu A (2003) Attenuation of dopamine transporter activity by a-synuclein Neurosci Lett 340, 189–192 12 Song YJ, Lundvig DM, Huang Y, Gai WP, Blumbergs PC, Hojrup P, Otzen D, Halliday GM & Jensen PH (2007) p25a relocalizes in oligodendroglia from myelin to cytoplasmic inclusions in multiple system atrophy Am J Pathol 171, 1291–1303 13 Hlavanda E, Kovacs J, Olah J, Orosz F, Medzihradszky KF & Ovadi J (2002) Brain-specific p25 protein binds to tubulin and microtubules and induces aberrant microtubule assemblies at substoichiometric concentrations Biochemistry 41, 8657–8664 14 Baker KG, Huang Y, McCann H, Gai WP, Jensen PH & Halliday GM (2006) P25a immunoreactive but a-synuclein immunonegative neuronal inclusions in multiple system atrophy Acta Neuropathol 111, 193–195 15 Jellinger KA (2006) P25a immunoreactivity in multiple system atrophy and Parkinson disease Acta Neuropathol 112, 112 16 Lindersson E, Lundvig D, Petersen C, Madsen P, Nyengaard JR, Hojrup P, Moos T, Otzen D, Gai WP, Blumbergs PC et al (2005) p25a stimulates a- synuclein aggregation and is co-localized with aggregated a-synuc- 504 17 18 19 20 21 22 23 24 25 26 27 28 lein in a-synucleinopathies J Biol Chem 280, 5703– 5715 Kragh CL, Lund LB, Febbraro F, Hansen HD, Gai WP, El-Agnaf O, Richter-Landsberg C & Jensen PH (2009) a-synuclein aggregation and Ser-129 phosphorylation-dependent cell death in oligodendroglial cells J Biol Chem 284, 10211–10222 Chen R, Tilley MR, Wei H, Zhou F, Zhou FM, Ching S, Quan N, Stephens RL, Hill ER, Nottoli T et al (2006) Abolished cocaine reward in mice with a cocaine-insensitive dopamine transporter Proc Natl Acad Sci USA 103, 9333–9338 Otzen DE, Lundvig DM, Wimmer R, Nielsen LH, Pedersen JR & Jensen PH (2005) p25a is flexible but natively folded and binds tubulin with oligomeric stoichiometry Protein Sci 14, 1396–1409 Aramini JM, Rossi P, Shastry R, Nwosu C, Cunningham K, Xiao R, Liu J, Baran MC, Rajan PK, Acton TB et al (2008) Solution NMR structure of tubulin polymerization-promoting protein family member from Homo sapiens http://www.pdb.org/pdb/explore/ explore.do?structureId=2JRF Accessed September 16 2008 Kobayashi N, Koshiba S, Inoue M, Kigawa T & Yokoyama S (2005) Solution structure of mouse CGI-38 protein http://www.pdb.org/pdb/explore/explore.do? structureId=1WLM Monleon D, Chiang Y, Aramini JM, Swapna GV, Macapagal D, Gunsalus KC, Kim S, Szyperski T & Montelione GT (2004) Backbone 1H, 15N and 13C assignments for the 21 kDa Caenorhabditis elegans homologue of ‘brain-specific’ protein J Biomol NMR 28, 91–92 Wersinger C, Jeannotte A & Sidhu A (2006) Attenuation of the norepinephrine transporter activity and trafficking via interactions with a-synuclein Eur J Neurosci 24, 3141–3152 Wersinger C, Rusnak M & Sidhu A (2006) Modulation of the trafficking of the human serotonin transporter by human alpha-synuclein Eur J Neurosci 24, 55–64 Jensen PH, Nielsen MS, Jakes R, Dotti CG & Goedert M (1998) Binding of a-synuclein to brain vesicles is abolished by familial Parkinson’s disease mutation J Biol Chem 273, 26292–26294 Lotharius J & Brundin P (2002) Impaired dopamine storage resulting from a-synuclein mutations may contribute to the pathogenesis of Parkinson’s disease Hum Mol Genet 11, 2395–2407 Langston JW (1989) Mechanisms underlying neuronal degeneration in Parkinson’s disease: an experimental and theoretical treatise Mov Disord 4, S15–S25 Takahashi N, Miner LL, Sora I, Ujike H, Revay RS, Kostic V, Jackson-Lewis V, Przedborski S & Uhl GR (1997) VMAT2 knockout mice: heterozygotes display reduced amphetamine-conditioned reward, enhanced FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS A W Fjorback et al 29 30 31 32 33 34 amphetamine locomotion, and enhanced MPTP toxicity Proc Natl Acad Sci USA 94, 9938–9943 ´ ´ ´ ´ Kovacs GG, Laszlo L, Kovacs J, Jensen PH, ´ Lindersson E, Botond G, Molnar T, Perczel A, Hudecz F, Mezo G et al (2004) Natively unfolded tubulin polymerization promoting protein TPPP ⁄ p25 is a common marker of alpha-synucleinopathies Neurobiol Dis 17, 155–162 ´ Kovacs GG, Gelpi E, Lehotzky A, Hoftberger R, Erdei ă A, Budka H & Ovadi J (2007) The brain-specific protein TPPP ⁄ p25 in pathological protein deposits of neurodegenerative diseases Acta Neuropathol 113, 153– 161 Skjoerringe T, Lundvig DM, Jensen PH & Moos T (2006) P25a ⁄ Tubulin polymerization promoting protein expression by myelinating oligodendrocytes of the developing rat brain J Neurochem 99, 333–342 Acevedo K, Li R, Soo P, Suryadinata R, Sarcevic B, Valova VA, Graham ME, Robinson PJ & Bernard O (2007) The phosphorylation of p25 ⁄ TPPP by LIM kinase inhibits its ability to assemble microtubules Exp Cell Res 313, 4091–4106 Wersinger C, Vernier P & Sidhu A (2004) Trypsin disrupts the trafficking of the human dopamine transporter by a-synuclein and its A30P mutant Biochemistry 43, 1242–1253 Wersinger C & Sidhu A (2005) Disruption of the interaction of a-synuclein with microtubules enhances cell surface recruitment of the dopamine transporter Biochemistry 44, 13612–13624 p25a regulates dopamine transporter function ´ 35 Vincze O, Tokesi N, Olah J, Hlavanda E, Zotter A, ă Horvath I, Lehotzky A, Tirian L, Medzihradszky KF, ´ Kovacs J et al (2006) Tubulin polymerization promoting proteins (TPPPs): members of a new family with distinct structures and functions Biochemistry 45, 13818–13826 36 Gu HH, Ahn J, Caplan MJ, Blakely RD, Levey AI & Rudnick G (1996) Cell-specific sorting of biogenic amine transporters expressed in epithelial cells J Biol Chem 271, 18100–18106 37 Mortensen OV, Kristensen AS, Rudnick G & Wiborg O (1999) Molecular cloning, expression and characterization of a bovine serotonin transporter Brain Res Mol Brain Res 71, 120–126 38 Jensen LD, Vinther-Jensen T, Kahns S, Sundbye S & Jensen PH (2006) Cellular parkin mutants are soluble under non-stress conditions Neuroreport 17, 1205–1208 39 Kleinnijenhuis AJ, Hedegaard C, Lundvig D, Sundbye S, Issinger OG, Jensen ON & Jensen PH (2008) Identification of multiple post-translational modifications in the porcine brain specific p25a J Neurochem 106, 925–933 40 Kubo S, Kitami T, Noda S, Shimura H, Uchiyama Y, Askawa S, Minoshima S, Shimizu N, Mizuno Y & Hattori N (2001) Parkin is associated with cellular vesicles J Neurochem 78, 42–54 41 Larsen MB, Fjorback AW & Wiborg O (2006) The C-terminus is critical for the functional expression of the human serotonin transporter Biochemistry 45, 1331–1337 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 505 ... instability of DA at the neutral pH of the cytosol causes the formation of toxic aminochromes, and these species are not formed when DA is protonated at the acidic pH of the storage vesicles Factors... from the top of the gradient The isolated fractions were subjected to reducing SDS ⁄ PAGE and immunoblotting for detection of endogenous p25a (E -p25a) , BIP and synaptophysin BIP and synaptophysin... supplemented with 1% Triton X-100 and 1% SDS to A B Fig p25a binds to porcine brain vesicles (A) The wild-type and deletion constructs of p25a p25aDN, p25aDC and the p25a core, corresponding to residues